Format

Send to

Choose Destination
Cell Stem Cell. 2014 Nov 6;15(5):643-52. doi: 10.1016/j.stem.2014.10.004. Epub 2014 Nov 6.

Efficient ablation of genes in human hematopoietic stem and effector cells using CRISPR/Cas9.

Author information

1
Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA 02138, USA; Program in Cellular and Molecular Medicine, Division of Hematology/Oncology, Boston Children's Hospital, Boston, MA 02116, USA.
2
Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA 02138, USA; Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138, USA.
3
Molecular Neurogenetics Unit, Psychiatric and Neurodevelopmental Genetics Unit, Center for Human Genetic Research, Massachusetts General Hospital, Boston, MA 02114, USA.
4
Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA 02138, USA.
5
Ragon Institute of MGH, MIT and Harvard, Cambridge, MA 02139, USA.
6
Ragon Institute of MGH, MIT and Harvard, Cambridge, MA 02139, USA; Center for Immunology and Inflammatory Diseases, Massachusetts General Hospital and Harvard Medical School, Charlestown, MA 02129, USA.
7
Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA 02138, USA; Program in Cellular and Molecular Medicine, Division of Hematology/Oncology, Boston Children's Hospital, Boston, MA 02116, USA; Department of Pediatrics, Harvard Medical School, Boston, MA 02115, USA.
8
Institution for Experimental Medical Research, Immunology section, Lund University, 221 84, Lund, Sweden.
9
Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA 02138, USA; Harvard Stem Cell Institute, Cambridge, MA 02138, USA; Division of Cardiovascular Medicine, Brigham and Women's Hospital, Boston, MA 02115, USA; Broad Institute, Cambridge, MA 02142, USA.
10
Molecular Neurogenetics Unit, Psychiatric and Neurodevelopmental Genetics Unit, Center for Human Genetic Research, Massachusetts General Hospital, Boston, MA 02114, USA; Broad Institute, Cambridge, MA 02142, USA; Department of Neurology, Harvard Medical School, Boston, MA 02115, USA.
11
Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA 02138, USA; Program in Cellular and Molecular Medicine, Division of Hematology/Oncology, Boston Children's Hospital, Boston, MA 02116, USA; Department of Pediatrics, Harvard Medical School, Boston, MA 02115, USA; Harvard Stem Cell Institute, Cambridge, MA 02138, USA. Electronic address: derrick.rossi@childrens.harvard.edu.
12
Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA 02138, USA; Harvard Stem Cell Institute, Cambridge, MA 02138, USA; Broad Institute, Cambridge, MA 02142, USA; Center for Regenerative Medicine, Massachusetts General Hospital, Boston, MA 02114, USA. Electronic address: chad_cowan@harvard.edu.

Abstract

Genome editing via CRISPR/Cas9 has rapidly become the tool of choice by virtue of its efficacy and ease of use. However, CRISPR/Cas9-mediated genome editing in clinically relevant human somatic cells remains untested. Here, we report CRISPR/Cas9 targeting of two clinically relevant genes, B2M and CCR5, in primary human CD4+ T cells and CD34+ hematopoietic stem and progenitor cells (HSPCs). Use of single RNA guides led to highly efficient mutagenesis in HSPCs but not in T cells. A dual guide approach improved gene deletion efficacy in both cell types. HSPCs that had undergone genome editing with CRISPR/Cas9 retained multilineage potential. We examined predicted on- and off-target mutations via target capture sequencing in HSPCs and observed low levels of off-target mutagenesis at only one site. These results demonstrate that CRISPR/Cas9 can efficiently ablate genes in HSPCs with minimal off-target mutagenesis, which could have broad applicability for hematopoietic cell-based therapy.

PMID:
25517468
PMCID:
PMC4269831
DOI:
10.1016/j.stem.2014.10.004
[Indexed for MEDLINE]
Free PMC Article

Supplemental Content

Full text links

Icon for Elsevier Science Icon for PubMed Central
Loading ...
Support Center